I propose to continue a study of several tRNA's in E. coli, with the goal of describing the specificity of their interactions with other cellular components. I will rely heavily on genetic and biochemical techniques developed in this laboratory during the past two years to 1. isolate mutants based on impaired functionality, and 2. characterize these mutants with respect to their recognition by processing enzymes, aminoacyl-tRNA synthetases, ribosomes, and regulatory elements. The mutant tRNA's will be purified by hybridization to plasmid DNA carrying the structural gene and their nucleotide sequences determined. It is hoped that this study will define the tRNA structural domains involved in the various protein-nucleic acid interactions. Particular emphasis will be placed on those tRNA mutants which, as desceibed in this proposal, have been shown to affect the expression of their corresponding amino acid biosynthetic operon.